Summary/Abstract Chronic bacterial infections such as those afflicting wounds and the airways of people with cystic fibrosis (CF) burden millions in the US. Prolonged antibiotic treatment often fails to eradicate these infections. However, bacteria such as Pseudomonas aeruginosa are often sensitive to antibiotics when treated ex vivo, indicating that conditions at sites of infection produce bacterial tolerance phenotypes. Currently, it is not clear how tolerance is produced in vivo, although several ideas have been proposed such as biofilm formation or the presence of persister cells. Regardless of the mechanism, it is clear that in vivo conditions produce tolerance. Sites of chronic infection are often polymer-rich; CF airway secretions contain abundant mucin, DNA, and other polymers. Work described in this application focus on molecular crowding caused by polymers. For example, polymer-induced crowding can force bacteria to aggregate by a mechanism called depletion aggregation. Preliminary results indicate that polymer-induced crowding rapidly establishes tolerance to antibiotics, antimicrobial peptides, and membrane disruption by EDTA, and tolerance is produced, in part, by the activation of the SOS DNA damage response. However, it is not clear if bacterial aggregation is required for polymer- induced crowding to produce tolerance. The long-term goal of the candidate, Dr. Patrick R. Secor, is to establish an independent research program focused on how disease-relevant environmental conditions affect pathogenesis phenotypes. As a step to achieve this goal, the immediate career objective of Dr. Secor is to obtain an independent faculty position using the research proposed in this application as the foundation for his job applications. The overall research objective of this application is to understand the mechanisms by which polymer-induced crowding produces antibiotic tolerance in P. aeruginosa. The hypothesis is that polymer- induced crowding produces antibiotic tolerance independent of bacterial aggregation and that polymer-induced crowding produces tolerance by causing SOS-activating genotoxic stress and by inducing cell envelope modifications. To test these hypotheses, three specific aims are proposed. Aim 1 will determine if bacterial aggregation is required for polymer-induced crowding to produce tolerance. Aim 2 will investigate how the SOS response is initiated by polymer-induced crowding by examining genotoxic stress. Aim 3 will determine whether polymer-induced crowding induces cell envelope modifications that produce tolerance using mass spectrometry. These aims are expected to demonstrate mechanistically how crowded environments such as those found at sites of chronic infection produce tolerance. In addition, data generated by these aims are expected to provide preliminary data for a competitive R01 application within two years of this award. This application also proposes career development activities aimed at complimenting Dr. Secor's prior experience. Dr. Pradeep Singh, the postdoctoral advisor to Dr. Secor, is included as a scientific advisor as he is a respected microbiologist with a strong track record of producing successful independent academic scientists.